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1 CIRAD, UMR Peuplement végétaux et Bioagresseurs en Milieu Tropical CIRAD/Université de la Réunion, Ligne Paradis, 97410 Saint Pierre, Réunion Island, France
2 UMR de Pathologie Végétale, INRA, Station de Pathologie Végétale, 42 rue Georges Morel, BP 57, 49071 Beaucouzé Cedex, France
3 Department of Plant Pathology, University of Florida, PO Box 110680, Gainesville, FL 32611-0680, USA
4 UMR 385 ENSAM-INRA-CIRAD, Biologie et Génétique des Interactions Plante–Parasite, Avenue Agropolis, 34398 Montpellier Cedex 5, France
Correspondence
Olivier Pruvost
olivier.pruvost{at}cirad.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Published online ahead of print on 27 June 2003 as DOI 10.1099/ijs.0.02714-0.
The GenBank accession number for the partial 16S rDNA sequence of Xanthomonas axonopodis pv. allii CFBP 6369 is AY135649.
Primer/adaptor sequences, a dendrogram based on FAME analysis, Biolog results and correspondence analysis and a fuller version of Fig. 1
are available as supplementary material in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The first characterization of the pathogen was performed in Hawaii, where the disease appeared in 1975 (Alvarez et al., 1978). A disease inducing similar symptoms was reported in mainland USA in the 1940s and ascribed to ‘Xanthomonas striaformans’ (Thomas & Weinhold, 1953), but the biochemical characteristics of this species do not match those of the genus (Swings et al., 1993). BBO has subsequently been reported in many countries in the Americas, Asia and Africa (Isakeit et al., 2000; Kadota et al., 2000; Neto et al., 1987; Nunez et al., 2002; O'Garro & Paulraj, 1997; Roumagnac et al., 1997; Sanders et al., 2003; Schwartz & Otto, 2000; Serfontein, 2001; Trujillo & Hernandez, 1999).
The taxonomic position of strains of Xanthomonas species pathogenic to onion has never been determined, neither in the reclassification of Xanthomonas performed by Vauterin et al. (1995), nor in a recent study comparing amplified fragment length polymorphism (AFLP) analysis and repetitive extragenic palindromic PCR (rep-PCR) to DNA–DNA relatedness in the genus Xanthomonas (Rademaker, 2000; Rademaker et al., 2000). Bacteria recently isolated from Welsh onion in Japan were considered to be a novel pathovar of Xanthomonas, and the name Xanthomonas campestris pv. allii pv. nov. was proposed (Kadota et al., 2000). This classification was, however, based only on biochemical and physiological characteristics and pathogenicity tests with a range of host plants, which is insufficient for species definition according to the recommendations of Wayne et al. (1987) and Stackebrandt et al. (2002). Kadota et al. (2000) have shown that the bacterium is pathogenic to onion and Welsh onion, but non-pathogenic to chive, Chinese chive and hyacinth. However, strains isolated in Barbados were pathogenic to onion, chive, leek, garlic, shallot (Bowen et al., 1998), French bean, soybean, winged bean, field pea, moth bean and lima bean (O'Garro & Paulraj, 1997), whereas strains isolated in Hawaii were pathogenic to onion and Welsh onion, but not to bean (Alvarez et al., 1978).
Because of the partial characterization of the bacterium associated with BBO and the incomplete definition of its host range, the present study was initiated to characterize a large worldwide collection of BBO strains by a polyphasic approach based on 16S rRNA gene sequencing, DNA–DNA hybridization, analyses of carbon source utilization, fatty acid methyl esters and fluorescent amplified fragment length polymorphisms (FAFLPs) and pathogenicity tests. Based on the results obtained, the denomination Xanthomonas axonopodis pv. allii is proposed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Vauterin et al., 1995) and some additional Xanthomonas axonopodis pathovars were used in this study (Tables 1 and 2). Cultures were stored as lyophilized cells and/or in a freezer at -80 °C; they were checked for purity and grown routinely on YPGA (7 g yeast extract, 7 g peptone, 7 g glucose and 15 g agar l-1; 20 µg propiconazole ml-1; pH 7·2) at 28 °C, except Xanthomonas populi, which was grown at 19 °C. All strains included in this study were deposited in the Collection Française de Bactéries Phytopathogènes (CFBP, INRA Angers, France) and strain numbers used throughout are CFBP numbers, except where noted.
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). Onion was screened as a potential host of X. axonopodis pv. phaseoli, because beans have been reported to be a host of the xanthomonad that causes BBO (O'Garro & Paulraj, 1997). Onion was also inoculated with Xanthomonas hyacinthi, the only xanthomonad from the Liliaceae for which a taxonomic position has been clearly determined (Table 2). All bacterial suspensions used for pathogenicity tests were prepared in 0·01 M Tris/HCl buffer (Sigma; pH 7·2) and adjusted spectrophotometrically (Milton Roy Spectronic 601) to approximately 108 c.f.u. ml-1 (OD600 of 0·05). These suspensions were further diluted in Tris buffer to reach the bacterial densities mentioned below. The Allium species (ten plants per species and strain) were spray-inoculated as described previously (Roumagnac et al., 2000) using bacterial suspensions containing approximately 107 c.f.u. ml-1. Ten bean plants were inoculated per strain by finger-rubbing bacterial suspensions of approximately 105 c.f.u. ml-1 on all trifoliate leaves of each plant. After inoculation, plants were covered with a translucent plastic bag for 48 h. Four hyacinth plants were inoculated per strain by pricking the leaf with a needle and then infiltrating 0·5 ml bacterial suspensions containing approximately 105 c.f.u. ml-1. Negative controls consisting of four (hyacinth) or ten (bean and Allium spp.) individuals were inoculated similarly with sterile buffer in all experiments. All plants were placed in a growth chamber at a temperature of 30±1 °C, except for hyacinth (12 h per day at 24±1 °C and 12 h at 18±1 °C), under a relative humidity of 95±5 % and a photoperiod of 12 h. Symptom appearance and progress were observed periodically after inoculation for 2 weeks. All experiments were repeated once.
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. Pectinolytic activity and gelatin hydrolysis were respectively assayed according to Hildebrand (1971) and Lelliott et al. (1966).
Fatty acid methyl ester analysis.
Strains were grown on medium containing nutrient broth (Difco) and 0·8 % agar for 24 h at 28 °C. Cultures were then transferred to a medium containing trypticase soy broth (BBL) and 0·75 % agar and were grown at 28 °C for 24 h. Strains 1976, 2532 and 4691 required 48 h incubation. Strain 4924 exhibited minimal growth on nutrient agar and was grown exclusively on TSBA. X. populi was not included in the analysis due to its very poor growth on all media tested. Extraction of fatty acids was performed according to the procedure described in MIDI Technical Note 101 (http://www.midi-inc.com/media/pdfs/TechNote_101.pdf). Bacterial strains were identified using the Sherlock TSBA40 version 4.00 method and a library running with the MIDI system version 4.13 DOS edition software. The GC system consisted of an HP 5890 GC, an HP 7673A autosampler and an HP 3396 series II integrator. Fatty acids were separated on an Ultra2 cross-linked 5 % PHME siloxane 25 M column and detected with an FID. Hydrogen was used as the carrier gas. A dendrogram edited as described by Norman et al. (1997) was based on data from 32 BBO strains and 20 type strains of Xanthomonas species. Mean fatty acid profiles were generated using the library generation software.
Utilization of carbon sources.
Six BBO strains (6107, 6359, 6362, 6367, 6369 and 6383) and the type strains of 19 Xanthomonas species were characterized phenotypically by the Biolog GN microplate system. Xanthomonas fragariae and X. populi were not analysed because of their poor reactivity in GN microplates. Experiments were performed as described by Vauterin et al. (1995). Two microplates were inoculated per strain and incubated at 28 °C for 48 h. Plates were then scored visually for carbon source utilization and OD590 were determined for the 96 wells using a microplate reader (Biotek EL 320). OD data were analysed by correspondence analysis with the software ADE-4 (Thioulouse et al., 1997). Groups of strains were defined by the Ward clustering method (Ward, 1963) using the same software. Biolog GN data were also used for identification purposes using the Microlog 1 software (Biolog) release 3.50.
Molecular methods
Sequencing of the 16S rRNA gene.
Total DNAs of strains 6364, 6366, 6367 and 6369 were extracted by the CTAB method (Ausubel et al., 1991). Amplification of the 16S rRNA genes was performed by PCR as described by Nesme et al. (1995) using the specific primers FGPS6 (5'-GGAGAGTTAGATCTTGGCTCAG-3') and FGPS1509' (5'-AAGGAGGGGATCCAGCCGCA-3'). The amplified 16S rDNA fragments were purified with a QIAquick PCR purification kit (Qiagen) and cloned into the pGEM-T easy plasmid as recommended by the manufacturer (Promega). Sequence data were obtained by single-pass double-stranded analysis (Genome Express) using primers T7 and SP6, which flank the cloning region in the pGEM-T easy plasmid. Sequence data were compared with those of other xanthomonads (Hauben et al., 1997; Trébaol et al., 2000) by alignment using the CLUSTAL method of MEGALIGN (DNASTAR).
DNA–DNA hybridization.
DNA was extracted and purified according to Brenner et al. (1982). Native DNA of strain 6369 was labelled in vitro with tritium-labelled nucleotides by random priming using Megaprime DNA labelling system RPM 1604 (Amersham International). The S1 nuclease–trichloroacetic acid method used for DNA–DNA hybridization experiments was described by Crosa et al. (1973) and modified by Grimont et al. (1980). The reassociation temperature used was 70 °C. DNA–DNA hybridization experiments with tritium-labelled DNA of strain 6369 were performed at least twice.
Thermal stability of DNA reassociation.
For strains that had DNA–DNA hybridization values of between 55 and 70 % (Grimont, 1988) with strain 6369, the temperature at which 50 % reassociated DNA became hydrolysable by nuclease S1 (Tm) was determined using the method of Crosa et al. (1973). The 
Tm value corresponds to the Tm value of the homoduplex (strain 6369) minus the Tm value of the heteroduplex.
DNA base composition.
The G+C contents (mol%) of strains 6107, 6369, 6381 and 6385 were determined by the thermal denaturation method (Marmur & Doty, 1962) and calculated using the equation of Owen & Lapage (1976).
Fluorescent AFLP (FAFLP) analysis.
Six BBO strains (6107, 6359, 6362, 6367, 6369 and 6383), all type strains of Xanthomonas species, except that of X. populi, and 12 strains of X. axonopodis (Table 2) were submitted to FAFLP analysis. Genomic DNA was extracted and purified as described by Brenner et al. (1982) and/or using the DNeasy tissue kit (Qiagen). To test the reproducibility of the FAFLP technique, strain 6369 was used as a control in each independent AFLP experiment (15 runs). To test the consistency of the two extraction methods, DNA of strain 6369 extracted by both methods was submitted to the FAFLP protocol in three independent experiments. Two reaction mixtures were prepared for digestion and ligation. For the first mixture, 5 µl genomic DNA (2 ng µl-1) was mixed in a total volume of 15 µl with 3·6 U of each restriction enzyme EcoRI and MspI (Eurogentec), 2 µl 0·5 M NaCl, 0·2 µl 100x BSA solution (Biolabs), 1·5 µl 10x T4 ligase buffer (Biolabs), 1 µl 2 µM EcoRI adaptor (Cybergène) and 1 µl 20 µM MspI adaptor (Cybergène). The second mixture was prepared in a total volume of 5 µl, with 0·5 µl 10x T4 ligase buffer and 100 U T4 DNA ligase (Biolabs). The two reaction mixtures were combined and incubated for 3 h at 37 °C. After incubation, the reaction mixture was diluted tenfold with distilled water.
Two successive amplification reactions were then performed. Firstly, amplification was performed in 20 µl containing 4 µl diluted DNA from the restriction–ligation step, 2 µl 10x Goldstar buffer (Eurogentec), 5 mM MgCl2, 2 mM dNTPs, 0·25 µM each of EcoRI and MspI primer and 0·5 U Taq DNA polymerase (Goldstar red; Eurogentec). This pre-selective amplification consisted of incubation of the reaction mixture for 2 min at 72 °C followed by 20 cycles with the following cycle profile: a 20 s DNA denaturation step at 94 °C, 30 s annealing step at 56 °C and 2 min extension step at 72 °C. PCR products were then diluted tenfold with distilled water. Secondly, a selective amplification using a 5'-JOE-labelled selective EcoRI primer (EcoRI+G primer) and the unlabelled selective MspI primer (MspI+TG primer) was performed in a 20 µl reaction mixture containing 1·5 µl diluted pre-amplified DNA from the previous PCR, 2 µl 10x Goldstar buffer (Eurogentec), 2·5 mM MgCl2, 2 mM dNTPs, 50 nM labelled selective EcoRI primer, 250 nM selective MspI primer and 0·5 U Taq DNA polymerase (Goldstar red; Eurogentec). Sequences of the adaptors, primers and selective primers used in FAFLP are available in Supplementary Table A in IJSEM Online. The selective amplification consisted of a 2 min denaturation step at 94 °C, followed by 33 cycles of a DNA denaturation step at 94 °C for 20 s, a 30 s annealing step (see below) and a 2 min extension step at 72 °C. The annealing temperature was 66 °C for the first cycle and was then decreased by 1 °C per cycle for the next 9 cycles and was 56 °C for the last 23 cycles. A final extension step was performed at 60 °C for 30 min. Amplification reactions were performed in a PE-9700 thermocycler (Applied Biosystems). One microlitre from the selective amplification was added to 18·2 µl formamide and 0·3 µl GENESCAN TAMRA-500 ladder as an internal standard (Perkin Elmer) and denatured at 95 °C for 3 min. Samples were then submitted to capillary electrophoresis for 35 min (15 kV) in an ABI-310 Genetic Analyzer (Applied Biosystems) with performance optimized polymers POP-4. The FAFLP fingerprints were superimposed and compared visually using GENESCAN software (Applied Biosystems). The threshold for assigning a peak was set to 100 relative fluorescence. The presence and absence of fragments were scored in a binary matrix. A similarity matrix using the Jaccard coefficient was calculated (NTSYS 2.0) and an unweighted neighbour-joining tree (Gascuel, 1997) was built using DARWIN 4.0 software (CIRAD). The robustness of the tree was assessed by bootstrap analysis using the same software (1000 repeated samplings). Current genome mispairing (CGM) values were calculated as described by Mougel et al. (2002). CGMs are obtained from the mathematical transformation of Jaccard similarity coefficients into genetic distances, which takes into account the number of nucleotide sites that constrain the AFLP technique (Mougel et al., 2002).
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RESULTS |
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Phenotypic traits
Biochemical and physiological characteristics.
The BBO strains formed single colonies on YPGA medium after 3 days incubation at 28 °C. Colonies were yellow, convex, round and mucoid. Cells were Gram-negative rods. Strains were obligate aerobes and glucose was utilized oxidatively. Starch, gelatin, aesculin, cellulose and Tween 80 were hydrolysed. Proteolysis of milk occurred. Cytochrome c oxidase and urease were not detected. Indole was not produced and nitrates were not reduced. Fluorescent pigments were not produced. Strains produced H2S from cysteine and showed pectinolytic activity at pH values of 5·0, 7·0 and 8·5. Cells grew on YPGA medium containing up to 3 % NaCl.
Analysis of fatty acid methyl ester composition.
A dendrogram based on fatty acid methyl ester profiles of 32 BBO strains and 20 type strains of Xanthomonas species is available as Supplementary Fig. A in IJSEM Online. Thirty of the 32 BBO strains formed a cluster with a Euclidian distance of less than 6·3; strain groupings were unrelated to geographical origin or host. Type strains of Xanthomonas codiaei, Xanthomonas cynarae and Xanthomonas translucens also fell into this same cluster. Removing these strains from the dendrogram did not change the Euclidian distance between the onion strains (data not shown). There were two outliers (6107 and 6108 from Japan), with a Euclidian distance of approximately 9·8.
The onion strains and the two outliers each had over 20 fatty acids (data not shown). In both groups, three fatty acids, 15 : 0 iso, 15 : 0 anteiso and summed feature 3 (16 : 1 
7c/15 iso 2-OH) accounted for >50 % of each profile. Beyond that, the relative occurrence of other fatty acids differed in the two profiles (Table 3).
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According to the correspondence analysis, the first three principal components respectively accounted for 38, 13 and 11 % of total variance. Ward clustering analysis revealed the presence of four groups of strains. Superimposition of groups on the graph showing the first two components allowed discriminating carbon sources to be easily identified. The six BBO strains studied clustered in group 1, together with Xanthomonas sacchari, Xanthomonas arboricola pv. juglandis and Xanthomonas pisi; X. campestris pv. campestris, X. codiaei, Xanthomonas melonis and Xanthomonas cassavae formed group 2. Xanthomonas albilineans was the only species of group 3; and group 4 included the other 11 type strains of Xanthomonas species. The most characteristic substrates were D-saccharic acid for group 2 and D-mannitol and D-gluconic acid for group 3. Strains that belonged to groups 1 and 4 did not utilize these three carbon sources. Group 1 strains utilized cellobiose, D-galactose, gentiobiose, lactulose and 
-ketoglutaric acid, whereas strains of group 4 did not. Within group 1, BBO strains could be differentiated from X. sacchari by the utilization of quinic acid, from X. arboricola pv. juglandis by the utilization of dextrin, glycogen and maltose and from X. pisi by the utilization of cis-aconitic acid.
Molecular characterization
Sequencing of the 16S rRNA gene.
A fragment of 1499 bp corresponding to positions 28–1529 of the 16S rDNA of Escherichia coli was amplified by PCR and sequenced for each of the four BBO strains. The 16S rDNA sequences of the four BBO strains analysed were 100 % identical, but this sequence differed by at least one base from the equivalent sequence of each of the 21 species of Xanthomonas studied. The 16S rDNA sequence of the BBO strains showed similarities to sequences of the other xanthomonads ranging from 96·7 % (X. hyacinthi and X. translucens) to more than 99 % (Xanthomonas vasicola, X. arboricola, Xanthomonas hortorum, X. campestris, X. cynarae, Xanthomonas oryzae, X. pisi, X. cassavae, Xanthomonas vesicatoria, Xanthomonas cucurbitae, Xanthomonas bromi, X. codiaei, X. populi, X. fragariae and X. axonopodis).
DNA–DNA hybridization and Tm values.
DNA–DNA hybridization values between strain 6369 and the other BBO strains were higher than 70 % (Table 1). In contrast, DNA–DNA hybridization values between strain 6369 and the 21 type strains of Xanthomonas species and the type strains of three pathovars of X. axonopodis were lower than 70 % (Table 2). The percentage reassociation of strain 6369 was 55 % with X. axonopodis pv. axonopodis strain 4924 and 11–49 % with the other 20 type strains of Xanthomonas. The pathotype strains of three pathovars of X. axonopodis (pv. manihotis, pv. begoniae and pv. phaseoli) were 65–70 % related to strain 6369. The Tm values were 0·2 °C between strains 6369 and 2534 (pv. phaseoli) and 0·5 °C between strains 6369 and 4924 (pv. axonopodis).
DNA base composition.
The DNA G+C contents of strains 6107, 6369, 6381 and 6385 were respectively 62·7, 62·7, 62·3 and 62·3 mol%.
FAFLP analysis.
Data derived from 15 different runs performed with strain 6369 allowed non-reproducible band positions to be identified; these were not considered for band scoring of all runs and strains. The composition of FAFLP patterns was not influenced by the DNA extraction method and the different runs differed only by one or two bands among 43 bands. The high reproducibility of FAFLP was further supported by maximal bootstrap values when data from DNA samples extracted by both techniques were included in the data matrix (results not shown).
A total of 147 fragments was generated and all these fragments were polymorphic. FAFLP analysis generated 62 fragments for the BBO strains; 63 % of these fragments were polymorphic. All BBO strains clustered with pathotype strains of X. axonopodis pv. vesicatoria, pv. citrumelo and pv. alfalfae (Fig. 1). A fuller phylogenetic tree is available as Supplementary Fig. C in IJSEM Online.
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CGM values between strains belonging to different Xanthomonas species ranged from 6·0 (between X. oryzae 2532T and X. axonopodis pv. alfalfae 3836) to 13·8 % (between X. fragariae 2157T and X. axonopodis pv. begoniae 2524). The mean CGM values were 3·6±0·6 % between BBO strains and 5·5±1·5 % between strains of X. axonopodis. Comparison of CGMs and DNA–DNA reassociation values showed that genome mispairing was negatively and linearly correlated to DNA–DNA hybridization values (r2=0·80).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The species allocation made by these authors, based solely on a biochemical and physiological characterization, does not meet the recommendations of Wayne et al. (1987) and Stackebrandt et al. (2002). Our study was performed to assess the accurate taxonomic position of the bacterium. To achieve this, a worldwide collection of strains of xanthomonads pathogenic to Allium species was examined by appropriate methods recommended for the taxonomic description of bacterial species (Stackebrandt et al., 2002). Moreover, international standards for naming pathovars of phytopathogenic bacteria were also used (Dye et al., 1980).
In accordance with the results of phenotypic characterization, 16S rDNA sequence similarities indicated that the BBO strains belong to the genus Xanthomonas and should be added to the X. campestris core described previously by Hauben et al. (1997). However, 16S rDNA sequence analysis is known to be limited in terms of taxonomic resolution at the species level for members of the genus Xanthomonas (Moore et al., 1997).
Taxonomic relationships at the species level were assessed by DNA–DNA hybridization, thermal stability of DNA reassociation and FAFLP analysis. All BBO strains belonged to a single genomospecies, as indicated by DNA reassociation values with BBO strain 6369, which were 74–100 %. Several reports indicated that xanthomonads associated with a single disease phenotype can belong to more than one Xanthomonas genomospecies (Jones et al., 2000; Vauterin et al., 1995).
The type strains of Xanthomonas species were only 11–55 % related to strain 6369. The most closely related type strain was X. axonopodis (with 55 % DNA reassociation). This is below the 70 % threshold allowed for species definition (Wayne et al., 1987). However, three other pathovars belonging to X. axonopodis included in our study showed 65–70 % DNA relatedness to strain 6369. The close relationship between the BBO strains and X. axonopodis was confirmed by the low Tm values (0·2 and 0·5 °C) obtained with strains 2534 (X. axonopodis pv. phaseoli) and 4924 (X. axonopodis pv. axonopodis), respectively. These values, which are much lower than the 5 °C Tm threshold (Wayne et al., 1987), indicate that the BBO causal agent should be included in the species X. axonopodis [i.e. group 9 defined by Vauterin et al. (1995)]. Other xanthomonads (e.g. pv. coracanae, pv. phaseoli, pv. sesbaniae, pv. vitians) with less than 70 % DNA relatedness to X. axonopodis have previously been allocated to this species (Trébaol et al., 2000; Vauterin et al., 1995). Thus, our data further confirm that X. axonopodis has a wider internal genetic variability than is commonly found in Xanthomonas species. Results from FAFLP analysis further confirmed that BBO strains were more similar to X. axonopodis than to any other Xanthomonas species. The unweighted neighbour-joining tree inferred from FAFLP patterns showed that BBO strains clustered with three pathovars of X. axonopodis, pv. vesicatoria, pv. citrumelo and pv. alfalfae. This is in full agreement with the results of Janssen et al. (1996), who used a different set of restriction enzymes and selective primers for AFLP and showed that X. axonopodis formed two clusters. One of them, cluster I, consisted of three subdivisions, , 
and 
; the subdivision contained the two pathovars alfalfae and citrumelo (pv. vesicatoria was not included in their study) with a linkage level of 71·0±3·7 %. In our case, the linkage level, calculated with the same index as Janssen et al. (1996), between strains of the ‘alfalfae–citrumelo–vesicatoria–BBO’ group was 67·6 % (data not shown). Similarly, Rademaker et al. (2000) analysed 80 AFLP and 178 rep-PCR (rep-, BOX- and ERIC-PCR) fingerprints of Xanthomonas strains. X. axonopodis consisted of six genetic clusters. Pathovars alfalfae, citrumelo and vesicatoria all belonged to the same group, designated 9.2 (Rademaker, 2000). In addition to the results of Rademaker et al. (2000), our study indicates that AFLP data are well correlated with DNA–DNA hybridization data at the species level and, therefore, the allocation of BBO strains to the species X. axonopodis was confirmed.
The CGM value provides an accurate measure of the genetic distance between genomes (Mougel et al., 2002). From data obtained on Agrobacterium (Mougel et al., 2002), a CGM of 13·6 % corresponded to the usual 70 % hybridization threshold above which strains are considered to belong to a single species (Wayne et al., 1987). In our study, the maximal CGM value obtained between Xanthomonas type strains and the BBO strain used as a probe for DNA–DNA hybridization was 9·1 %, indicating that AFLP profiles of distinct species of Xanthomonas may be closer than those of distinct species of Agrobacterium. Furthermore, a highly significant linear relationship between CGM and DNA–DNA hybridization values was found over the whole range of values. Our data further confirm the conclusions of Mougel et al. (2002), who stated that the correspondence between CGM and the threshold of 70 % DNA relatedness must be established with care for different bacterial genera.
Correspondence analysis was used to compare metabolic fingerprints obtained from the type strains of Xanthomonas species and six BBO strains. This statistical method has already been used for phenotypic characterization of bacterial populations, and its major advantages for eliminating bias related to the Biolog technique have already been discussed (Frey et al., 1997; Garland, 1996). BBO strains grouped with X. sacchari, X. arboricola pv. juglandis and X. pisi. Correspondence analysis resulted in determination of the most useful carbon sources for identification purposes. However, metabolic fingerprints of type strains of Xanthomonas species obtained in this study differed from those reported by Trébaol et al. (2000) and Vauterin et al. (1995), which also differed from each other. Scortichini & Rossi (1995) reported that subculturing influenced carbohydrate utilization profiles of X. campestris pv. campestris. Recently, Riley et al. (2001) reported that carbon utilization profiles were variable among Ralstonia populations evolved in defined environments for 1000 generations. Most of the type strains of Xanthomonas species were isolated more than 30 years ago, and changes in phenotypic traits could have occurred. Such changes could be related to the mode and duration of conservation, the number of successive strain subcultures and occurrence of stresses during exchanges of cultures, etc. The lack of stability of phenotypic traits probably represents a bias when xanthomonad strains from culture collections are compared, and emphasizes the difficulty of comparing metabolic fingerprints obtained in a laboratory with previously published information. As a consequence, the lack of reproducibility questions the power of the Biolog technique for locating unknown xanthomonads in the classification described by Vauterin et al. (1995).
Although data from the fatty acid analysis were not in agreement with results from DNA–DNA hybridization and FAFLP, this analysis was useful in showing that most of the BBO strains form a tight group. All but two BBO strains formed a cluster with a Euclidian distance of less than 6·3. According to MIDI, strains with a Euclidian distance of approximately less than 6·0 are considered to be the same subspecies or biotype. The two strains originating from Japan (6107 and 6108) were outliers. Strain 6107 has been chosen by Kadota et al. (2000) as the pathotype strain. Based on fatty acid methyl ester analysis, this strain is not typical of the pathovar. Therefore, strain 6369, which was shown to be typical of the pathovar by all techniques used in this study, was chosen as the probe for DNA–DNA hybridization. It is proposed that strain 6369 should be established as the neopathotype strain for pv. allii.
The present study was also initiated to clarify the host range of BBO strains. All BBO strains were pathogenic to all Allium species assayed. This result is in agreement with those reported by Bowen et al. (1998), but diverged from those described by Kadota et al. (2000). The latter authors determined that BBO strains were not pathogenic to chive using Allium schoenoprasum L. var. foliosum. In the present work, A. schoenoprasum L. cv. Civette, one of the most common cultivars worldwide, was used. A. schoenoprasum L. grows in a variety of habitats, and many local ecotypes have arisen (Poulsen, 1990). This could explain why BBO strains are pathogenic to A. schoenoprasum L. but not to the variant foliosum. O'Garro & Paulraj (1997) listed bean and other plant species of the family Fabaceae as hosts of BBO strains in Barbados. In the present study, bean leaves inoculated with BBO strains (including isolates from Barbados) sometimes exhibited small, water-soaked lesions that remained for a few days before turning necrotic, without further expansion of the lesions (data not shown). Our results are consistent with those reported by Alvarez et al. (1978). Furthermore, several xanthomonads were reported to induce small, water-soaked lesions when inoculated onto bean (Schnathorst, 1966). Such lesions, which clearly differed from those observed during the compatible interaction between X. axonopodis pv. phaseoli and bean, were regarded as non-host interactions. Based on all the above results, the name Xanthomonas axonopodis pv. allii is proposed as the causal agent of BBO.
Description of Xanthomonas axonopodis pv. allii
Xanthomonas axonopodis pv. allii [al'li.i. N.L. adj. allii from Allium, the genus of onion (Allium cepa L.), Welsh onion (Allium fistulosum L.) and garlic (Allium sativum L.)].
Gram-negative rods, motile with one polar flagellum; obligate aerobes with oxidative metabolism of glucose and catalase-positive. Does not produce cytochrome c oxidase, nitrate reductase, arginine dihydrolase, urease, acetoin or indole. Colonies grown on YPGA after 2–3 days at 28 °C are yellow, convex, round and mucoid. Hydrolyses gelatin, casein, aesculin, starch, cellulose and Tween 80, produces H2S from cysteine and shows pectinolytic activity at pH 5·0, 7·0 and 8·5. Cells tolerate up to 3 % NaCl and grow on YPGA at 35 °C but not at 40 °C. Fluorescent pigments are not observed. Produces acid from xylose and ribose, but not from melezitose on ARJ medium. Metabolic activity (as assessed by Biolog GN microplates) is shown on the following carbon substrates: dextrin, glycogen, Tween 40, N-acetyl-D-glucosamine, cellobiose, D-fructose, D-galactose, gentiobiose, -D-glucose, lactulose, maltose, D-mannose, D-psicose, sucrose, D-trehalose, methyl pyruvate, monomethyl succinate, cis-aconitic acid, -ketobutyric acid, -ketoglutaric acid, malonic acid, succinic acid, bromosuccinic acid, alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-glutamic acid, glycyl L-glutamic acid, L-proline, L-serine and L-threonine. The following carbon substrates are not utilized: -cyclodextrin, N-acetyl-D-galactosamine, adonitol, L-arabinose, D-arabitol, i-erythritol, m-inositol, -D-lactose, D-mannitol, methyl -D-glucoside, L-rhamnose, D-sorbitol, xylitol, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, -hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, quinic acid, D-saccharic acid, sebacic acid, glucuronamide, L-histidine, L-ornithine, L-phenylalanine, L-pyroglutamic acid, D-serine, DL-carnitine, -aminobutyric acid, urocanic acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol and glucose 6-phosphate.
Induces typical BBO symptoms on leaves of onion (Allium cepa L.), garlic (Allium sativum L.), chive (Allium schoenoprasum L.), leek (Allium porrum L.), shallot (Allium cepa var. ascalonicum Backer) and Welsh onion (Allium fistulosum L.). Characteristic leaf lesions start as lenticular, water-soaked spots, which extend and eventually coalesce. Lesions progress into dry, chlorotic spots with tissue collapse and holes. Severe infections induce leaf dieback, resulting in a reduction in bulb size. Not pathogenic to Allium schoenoprasum L. var. foliosum Rgl., Chinese chive (Allium tuberosum Rottler), hyacinth (Hyacinthus orientalis L.) or bean (Phaseolus vulgaris L.). The DNA G+C content is 62·3–62·7 mol%. The proposed neopathotype strain is CFBP 6369=LMG 21894.
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